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by the Heavy Metal Cadmium
Department of Biochemistry and Molecular Biology (A.S., M.B.M.)
Vincent T. Lombardi Cancer Center Georgetown University
Washington, DC, 20007
Department of Molecular and Integrative
Physiology (B.S.K.) University of Illinois Urbana, Illinois
61801
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER-),
the ability of cadmium to bind to and activate wild-type and various
mutants of ER- was examined. When tested in transient cotransfection
assays in COS-1 cells, cadmium concentrations as low as
10-11 M activated
ER-. Scatchard analysis employing either purified human recombinant
ER- or extracts from ER-containing MCF-7 cells demonstrated that
109Cd binds to the ER with an equilibrium
dissociation constant of approximately 4 to 5 x
10-10 M. Cadmium also
blocks the binding of estradiol to ER- in a noncompetitive manner
(Ki = 2.96 x
10-10 M), suggesting
that the heavy metal interacts with the hormone-binding domain of the
receptor. To study the role of the hormone-binding domain in cadmium
activation, COS-1 cells were transiently cotransfected with GAL-ER, a
chimeric receptor containing the DNA-binding domain of the
transcription factor GAL4 and the hormone-binding domain of ER-, and
a GAL4-responsive reporter gene. Treatment of the transfected cells
with either 10-6 M
cadmium or 10-9 M
estradiol resulted in a 4-fold increase in reporter gene activity. The
effect of cadmium on the chimeric receptor was blocked by the
antiestrogen, ICI-164,384, suggesting that cadmium activates ER-
through an interaction with the hormone-binding domain of the receptor.
Transfection and binding assays with ER- mutants identified C381,
C447, E523, H524, and D538 as possible interaction sites of cadmium
with the hormone-binding domain of ER-. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Because the estrogen receptor (ER) is a critical mediator of growth,
molecules that can bind to and activate the ER can potentially increase
the risk of breast cancer. A number of natural and man-made chemicals
in the environment possess estrogenic activity and, therefore, may pose
a health risk. Data presented in this paper and in a previously
published study from this laboratory (10) suggest that the heavy metal,
cadmium, is a new environmental estrogen. In that study, cadmium was
shown to mimic the effects of estradiol in the estrogen-responsive
breast cancer cell line, MCF-7. Treatment with cadmium resulted in an
increase in cell growth, an increase in the steady state levels of
progesterone receptor, pS2, and cathepsin D, and a decrease in the
steady state level of estrogen receptor- (ER-). The changes in
steady state levels of protein and mRNA of these genes were due to
changes in transcription that were blocked by the antiestrogen,
ICI-164,384. Transfection assays also demonstrated that the effects of
cadmium were mediated by ER-.
The goal of the present study was to gain insight into the mechanism by
which cadmium activated ER-. To achieve this goal, the ability of
cadmium to bind to and activate wild-type ER and mutant forms of the
receptor was investigated. The results presented herein demonstrate
that low concentrations of cadmium activate ER- through an
interaction with the hormone-binding domain of the receptor. The metal
binds with high affinity and blocks estradiol binding to the receptor.
The interaction of cadmium with the receptor appears to involve several
amino acids in the hormone-binding pocket of the receptor, suggesting
that the metal may form a coordination complex with the hormone-binding
domain and thereby activate the receptor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gene and an
estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter
construct under the control of the mouse mammary tumor virus (MMTV)
promoter, in which the glucocorticoid response element was replaced
with two estrogen response elements (11), were transiently
cotransfected into COS-1 cells. After transfection, the cells were
treated for 24 h with either CdCl2
(10-12 to 10-6
M) or estradiol (10-9 and
10-8 M). The amount of
chloramphenicol acetyltransferase (CAT) activity was then measured,
expressed as percent conversion, and normalized to the amount of
ß-galactosidase activity (Fig. 1
). As
expected, treatment with estradiol resulted in an approximately 4-fold
increase in CAT activity. Treatment with 10-12
M to 10-6 M cadmium also
stimulated expression of CAT by approximately 2- to 6-fold, suggesting
that low doses of cadmium activated ER-.
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involved in activation by cadmium,
chimeric receptors containing the hormone-binding domain of either
ER- or the GR were employed (12). These chimeric receptors consist
of the DNA-binding domain of the yeast transcription factor GAL-4 and
the hormone-binding domain of either ER- (GAL-ER) or the GR
(GAL-GR). Stimulation of transcription by GAL-ER or GAL-GR from a
GAL-4-responsive reporter gene requires either estradiol or
dexamethasone, respectively. When the chimeric receptor GAL-ER and the
GAL-4-CAT reporter construct were transiently cotransfected into COS-1
cells and the cells were treated with 10-6
M cadmium or 10-9 M
estradiol there was an approximately 4-fold increase in CAT activity
(Fig. 2). Addition of the estrogen antagonist ICI 164,384 (5 x
10-7 M) blocked both the cadmium and
the estradiol-induced increase in transcription by 75%
(P = 0.0075) and 91%, respectively. Cadmium has been
shown to block the binding of dexamethasone to the GR (13). Since
cadmium interacts with the hormone-binding domain of the GR, GAL-GR was
employed as a control; the results are compared in Fig. 2. Cadmium and estradiol had no effect in
cells transfected with GAL-GR, while 10-9
M dexamethasone induced an approximately 4-fold
increase in CAT activity. The dexamethasone-induced increase was
inhibited by cadmium, demonstrating an antagonistic effect of the metal
on the GR. Taken together, these results suggest that cadmium interacts
with the hormone-binding domain of ER- to activate the receptor.
Although cadmium interacts with the hormone-binding domain of the GR,
it does not activate the GR.
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, the effects of the heavy metal on hormone binding were measured
using a single-dose ligand- binding assay. Extracts from MCF-7 cells
were treated with various concentrations of cadmium
(10-12 to 10-5
M) for 1 h. The ability of the ER to bind hormone was
then assayed by incubating the extract with 10-8
M [3H]estradiol in the presence or
absence of a 200-fold molar excess of diethylstilbestrol for 18 h
at 4 C. As shown in Fig. 3a, cadmium
blocked the binding of estradiol to the receptor. Hormone binding
decreased with increasing cadmium concentration. Hormone binding also
decreased with increasing preincubation time with the heavy metal (Fig. 3b, P = 0.0193). Maximal inhibition was seen after a
30-min preincubation with 10-6
M cadmium. In contrast to cadmium, preincubation
with high concentrations of zinc (10-6 to
10-4 M
ZnSO4) for 1 h did not influence hormone
binding to ER- (Fig. 3b and data not shown). These results
demonstrate a degree of specificity for the action of cadmium in
blocking the binding of estradiol to ER-.
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was incubated with
10-10, 10-9, or
10-8 M cadmium chloride and various
concentrations of [3H] estradiol
(10-12 to 10-7
M) in the presence or absence of a 200-fold molar excess of
diethylstilbestrol (DES). The affinity and binding capacity of the
receptor were determined according to the method of Scatchard (19)
(Fig. 4). In the absence of cadmium, estradiol bound to
the receptor with an equilibrium dissociation constant
(Kd) of 2.92 x 10-10
M (±0.34, n = 3). In the presence of cadmium
chloride, the dissociation constant of estradiol was unchanged, but the
number of binding sites decreased (Table 1). These data demonstrate that cadmium
competes with estradiol for binding to ER- in a noncompetitive
manner since it does not alter the binding affinity of hormone for the
receptor.
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. Recombinant human ER- was incubated with
various concentrations of 109Cd
(10-12 to 10-7
M) in the presence and absence of a 200-fold molar excess
of CdCl2; the results are shown in Fig. 5. Interestingly, cadmium bound to ER- with an
affinity similar to that of estradiol for the receptor. The
dissociation constant for cadmium binding is approximately 5 x
10-10 M (±1.5, n =
3). The binding of cadmium to the ER- in MCF-7 cells was also
measured and compared with the binding to recombinant receptor. A
similar dissociation constant was obtained (Kd =
4.5 x 10-10 M (±
2.1, n = 3, data not shown) when the binding of cadmium to ER-
was measured in whole-cell extracts. These results demonstrated that
there was no significant difference in the affinity of binding of
either cadmium or estradiol to the ER. Scatchard analysis also showed
that ER- contained approximately 1.1 cadmium-binding site.
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). After treatment of the cysteine
mutants with cadmium, there was an approximately 4-fold increase in CAT
activity of C417A and an approximately 8-fold increase with C530A. In
contrast to the effects observed with mutants C417A and C530A, cadmium
failed to activate the mutants C381A and C447A and the quadruple mutant
C381A-C417A-C447A-C530A, suggesting that cysteines C381 and
C447 are involved in cadmium activation of the ER. To demonstrate that
the mutation of cysteine to alanine did not interfere with the activity
of the receptor, the transiently transfected cells were treated with
estradiol. After hormone treatment, there was an approximately 4- to
5-fold increase in CAT activity with all of the mutants. These results
corroborate previous studies employing these mutants, which demonstrate
that mutation of cysteines to alanine in the hormone-binding domain
does not alter the ability of estradiol to transactivate the receptor.
To determine whether the cysteine mutants were capable of binding
cadmium, the mutants were expressed in COS-1 cells, and specific
binding of 109Cd was determined using a
whole-cell binding assay (Table 2). The
dissociation constants for binding of cadmium to the cysteine
mutants C381A and C447A were 5 x 10- 9
M and 3 x10-9 M,
respectively.
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). Cadmium did not activate mutants E523Q, E523A, H524A, or
D538N but activated E380Q, resulting in a 4.5-fold increase in CAT
activity; this suggests that glutamic acid E523, aspartic acid D538,
and histidine H524 may also play a role in the interaction of cadmium
with the ER. As expected, estradiol treatment of all four mutants
resulted in an approximately 4- to 5-fold increase in CAT activity,
suggesting that mutation of these amino acids did not interfere with
the activity of the ER. The ability of cadmium and estradiol to bind to
these mutants was also determined using a whole-cell binding assay
(Table 2). In contrast to the cysteine mutants, there was no detectable
binding of cadmium to mutants E523Q, H524A, and D538N. However, the
latter three mutants were capable of binding estradiol with high
affinity but at a lower affinity than the wild-type receptor. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. The ability of cadmium to activate the chimeric receptor is
blocked by an antiestrogen. Interaction of the metal with the receptor
also inhibits the binding of estradiol to the receptor. Mutational
analysis identified several amino acids as potential metal interaction
sites, suggesting that cadmium activates ER- through the formation
of a coordination complex within the hormone-binding domain of the
receptor.
The ER is a member of a superfamily of transcription-regulating
proteins that bind zinc. The binding of zinc to cysteines in the
DNA-binding domain of ER- results in the formation of a protein
motif referred to as a zinc finger (18). Other metals have been shown
to substitute for zinc in the zinc finger of ER- and to influence
the binding of the DNA-binding domain to an estrogen response element
(19). The replacement of zinc with either nickel or copper inhibits the
binding of the DNA-binding domain to an estrogen response element,
whereas replacement of zinc with either cadmium or cobalt has no effect
on specific DNA binding. In addition to the DNA-binding domain, metals
have been shown to bind to the hormone-binding domain of receptors and
to block binding of the ligand. In the case of ER-, calcium has been
shown to reversibly block the binding of estradiol (20). Interaction of
arsenite, cadmium, and selenite with cysteines in the hormone-binding
domain of the GR has also been shown to inhibit the binding of
dexamethasone to the receptor (13). Results presented in this study
suggest that the interaction of cadmium with ER- is similar to the
interaction of the metal with the GR, i.e. cadmium binds
with high affinity to the hormone-binding domain of ER- and blocks
the binding of estradiol. Interaction of cadmium with the ER also
appears to involve cysteines, specifically cysteines 381 and 447. In
addition to cysteines, the metal appears to interact with glutamic acid
523, histidine 524, and aspartic acid 538. Although the interaction of
cadmium with the ER results in activation of the receptor, this does
not appear to be the case for the GR.
Metals serve several different functions in proteins including
participation in catalytic reactions and stabilization of protein
structure. Through interactions with different amino acids, metals may
promote local folding, as in the case of the zinc finger, or assembly
of different regions of the protein into one domain (21). Cadmium is a
heavy metal that is capable of forming a coordination complex with a
number of different amino acids (reviewed in Ref. 22). It is found in
both tetrahedral and octahedral arrangements with the side chains of
cysteine, aspartic acid, histidine, and glutamic acid, and, to a lesser
extent, with serine. In addition to amino acid side chains, cadmium
interacts with the terminal nitrogen of serine, the peptide oxygen of
phenylalanine, lysine, arginine, asparagine, histidine, glutamine, and
water. Mutational analysis of ER- revealed several amino acids in
the hormone-binding domain (C381, C447, E523, H524, and D538) as
potential sites of interaction with the metal. The exact role of these
amino acids in interaction and activation of the ER by cadmium remains
unknown. It is possible that these amino acids participate directly in
the formation of the metal-binding site or indirectly in the
recruitment of cadmium to the binding site. However, the latter appears
unlikely. If the function of these amino acids is to recruit the metal
to its binding site, then mutation of any one of these amino acids
would not alter cadmium binding and activation of the receptor.
Mutation of either E523, H524, or D538 resulted in the complete loss of
binding and activation, suggesting that these amino acids play a more
integral role in the interaction and activation of the receptor than
recruitment. When the cysteines were mutated to alanines, the ability
of cadmium to bind to the receptor was not altered, but the ability of
cadmium to transactivate the receptor was lost, suggesting that the
interaction of the metal with these cysteines influences the formation
of an active conformation of the receptor.
Similar to other receptors (23, 24, 25, 26, 27, 28, 29), the hormone-binding domain of the
ER contains 12 -helices (H1–H12) folded into a three-layered
antiparallel -helical sandwich. The central core layer contains
three - helices (H5/6, H9, and H10) sandwiched between two
additional layers of helices composed of H1–4, H7, H8, and H11. The
central core of the hormone-binding domain is flanked by helix H12. It
has been proposed that the hormone-binding domain functions in a manner
similar to a mouse trap (23). Upon binding, the ligand induces a
conformational change resulting in the formation of a salt bridge
between H4 and H12, repositioning helix H12 over the central core and
consequently entrapping the hormone. Ultimately, the repositioning of
helix H12 results in the formation of a transcriptionally active
receptor. The amino acids identified as playing a role in the
interaction of cadmium with ER- are located on helices H4, H8, and
H11 and at the interface of the loop and H12. Cysteines 381 and 447 are
located on helices H4 and H8, respectively. Glutamic acid 523 and
histidine 524 are located on helix H11 and are in close proximity to
the ligand. Aspartic acid 538 is located at the loop-H12 interface and
is also reasonably close to the ligand. It is possible that interaction
of cadmium with these amino acids brings different helices of the
hormone- binding domain into close proximity. The interaction of
cadmium with these amino acids may mimic the effects of estradiol by
repositioning H12 similar to the repositioning observed upon hormone
binding. This model remains to be tested. It also remains to be
determined whether cadmium interacts directly or indirectly with these
amino acids through water molecules.
Cadmium is a heavy metal with no known physiological function. Human exposure to the metal occurs primarily through dietary sources, cigarette smoking, and, to a lesser degree, drinking water (30, 31). In newborns, the amount of cadmium found in the body is negligible, but by age 30, the body burden may reach 30 mg. Cadmium has a biological half-life estimated to range from 10 to 30 yr (32), which may account for the significant accumulation of the metal in the body. In nonsmokers, the kidney concentration of cadmium is approximately 15–20 µg/g wet weight, while in smokers, the concentration doubles to 30–40 µg/g wet weight. Interestingly, the human mammary gland, an estrogen target organ, also contains high concentrations of cadmium (31 µg/g) (33) suggesting that the metal may be a potential risk factor for breast cancer.
In this study, we provide evidence that the heavy metal cadmium is an
environmental estrogen. At low concentrations, the metal mimics the
effects of estradiol in transient transfection assays. It binds with
high affinity to the hormone-binding domain of ER- and blocks the
binding of estradiol to the receptor. Binding to the ER appears to
involve several amino acids, suggesting that cadmium activates the
receptor through the formation of a coordination complex with specific
residues in the hormone-binding domain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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expression vector (wild type or mutant, as described below), 75 µg of
a reporter construct, 6 µg of ß-galactosidase, and salmon sperm
carrier DNA. Sixteen to 18 h after transfection, the precipitate
was washed off, and the cells were replenished with phenol red-free
IMEM containing 10% CCS in the presence or absence of
10-9 M estradiol or
10-6 M cadmium chloride. The cells
were harvested 24 h later and CAT activity was measured as
described previously (10). CAT activity was expressed as the percent
conversion of chloramphenicol to its acetylated forms and was
normalized to the activity of ß-galactosidase. The increase in CAT
activity in response to treatment is expressed relative to untreated
controls. Statistical significance was determined by Student’s
t test.
Expression vectors for the wild-type ER- (pRER-wt) and the amino
acid mutants (C381A, C417A, C447A, C530A, E523A, D538N, H524A) are
described elsewhere (14, 15, 16, 17). For these transient transfection assays,
the estrogen-responsive reporter construct, pbCAT-(S)MERE (11), was
obtained from Dr. D. El Ashry (Lombardi Cancer Center, Georgetown
University, Washington, DC). The chimeric receptors, GAL-ER and GAL-GR,
and the reporter plasmid 17M2GCAT are also described elsewhere
(12).
ER Binding Assays
The ability of cadmium to block estradiol binding to the ER-
was determined in cell extracts from MCF-7 cells that were maintained
in phenol red-free IMEM containing 5% CCS for 2 days. After 2 days in
estrogen-depleted medium, the cells were lysed by sonication in a
high-salt buffer containing 10 mM Tris, pH 7.5, 1.5
mM EDTA, 5 mM sodium molybdate, 0.4
M KCl, 1 mM monothioglycerol, 2 mM
leupeptin. The homogenate was incubated on ice for 30 min and
centrifuged at 100,000 x g for 1 h at 4 C (35).
The protein concentration of the cell extract was determined by the
Bradford method. Unless indicated otherwise, cell extracts were
preincubated on ice for 60 min with various concentrations of
CdCl2 (10-12 to
10-5 M).
[3H]Estradiol, 10-8
M, was then added in the presence and absence of
a 200-fold molar excess of diethylstilbestrol (DES) and incubated at 4
C for 16–18 h. Free steroid was removed by the addition of 5%
dextran-coated charcoal. The amount of radioactivity was measured by
scintillation counting. Specifically bound complexes were calculated by
subtracting nonspecific binding from total binding.
The ability of cadmium to bind to the ER was determined using
recombinant human ER- (PanVera Corp., Madison, WI) and MCF-7 cell
extracts. Recombinant ER- (4 x 10- 9
M) was incubated with various concentrations of
109Cd (10-12 to
10-7 M, specific activity 90
µCi/µg cadmium chloride, Amersham Pharmacia Biotech,
Piscataway, NJ) in the presence and absence of a 200-fold molar excess
of CdCl2 for 16–18 h at 4 C. Free radioactivity
was removed by adding 5% dextran-coated charcoal. In the whole-cell
binding assay, 5 x 105 MCF-7 cells were
plated into six-well dishes in IMEM containing 5% CCS. The medium was
subsequently replaced with phenol red-free IMEM containing 5% CCS.
After 2 days in estrogen-depleted medium, the cells were incubated for
16–18 h at 4 C with various concentrations of
109Cd (10-12 to
10-7 M) in the presence and absence
of a 200-fold molar excess of CdCl2. Free
radioactivity was removed by washing. The cells were disrupted by four
freeze-thaw cycles, and the amount of 109Cd bound
to the ER was measured by scintillation counting. The data were
analyzed by the method of Scatchard (36). To determine whether cadmium
alters the binding affinity of estradiol for the ER, recombinant human
ER- was incubated with 10-10,
10-9, or 10-8
M cadmium and various concentrations of
[3H] estradiol (10-12 to
10-7 M) in the presence or absence
of a 200-fold molar excess of DES. The assay conditions and data
analysis are described above.
To measure the binding of cadmium and estradiol to ER mutants, COS-1
cells were plated into six-well dishes and transiently transfected with
either wild-type or mutant ER-. The transfected cells were incubated
at 4 C for 16 h with various concentrations of either
109Cd (10-12 to
10-7 M) or
[3H]estradiol (10-12 to
10-7 M) in the presence or absence
of a 200-fold molar excess of CdCl2 or DES,
respectively. Free radioactivity was removed by aspiration, and the
cells were disrupted by four freeze-thaw cycles. The amount of bound
radioactivity was quantitated by scintillation spectrophotometry, and
the data were analyzed as described above.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant CA-70708 (to M.B.M.), Cancer Research Foundation of America (to A.S.), The Susan G. Komen Foundation (to A.S.), and NIH Grant CA-18119 (to B.S.K.).
Received for publication July 13, 1999. Revision received December 28, 1999. Accepted for publication January 5, 2000.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M. B. Martin, R. Reiter, T. Pham, Y. R. Avellanet, J. Camara, M. Lahm, E. Pentecost, K. Pratap, B. A. Gilmore, S. Divekar, et al. Estrogen-Like Activity of Metals in Mcf-7 Breast Cancer Cells Endocrinology, June 1, 2003; 144(6): 2425 - 2436. [Abstract] [Full Text] [PDF]
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 G. E. Stoica, T. F. Franke, A. Wellstein, F. Czubayko, H.-J. List, R. Reiter, E. Morgan, M. B. Martin, and A. Stoica Estradiol Rapidly Activates Akt via the ErbB2 Signaling Pathway Mol. Endocrinol., May 1, 2003; 17(5): 818 - 830. [Abstract] [Full Text] [PDF]
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M. B. Martin, H. J. Voeller, E. P. Gelmann, J. Lu, E.-G. Stoica, E. J. Hebert, R. Reiter, B. Singh, M. Danielsen, E. Pentecost, et al. Role of Cadmium in the Regulation of AR Gene Expression and Activity Endocrinology, January 1, 2002; 143(1): 263 - 275. [Abstract] [Full Text] [PDF]
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, Control; •,
10-10 M cadmium; 
, 10-9
M cadmium; 
, 10-8 M cadmium. B,
Saturation curves of the [3H]estradiol binding to ER-
, Control;
, estradiol;